Optimization of apolipoprotein-B-100 sequence coverage by liquid chromatography-tandem mass spectrometry for the future study of its posttranslational modifications |
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Authors: | Delporte Cédric Van Antwerpen Pierre Zouaoui Boudjeltia Karim Noyon Caroline Abts Frédéric Métral Frédéric Vanhamme Luc Reyé Florence Rousseau Alexandre Vanhaeverbeek Michel Ducobu Jean Nève Jean |
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Affiliation: | aLaboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium;bAnalytical Platform of the Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium;cLaboratory of Experimental Medicine (ULB 222 Unit), CHU de Charleroi, A. Vesale Hospital, Université Libre de Bruxelles, Montigny-le-Tilleul, Belgium;dAgilent Technologies, Diegem, Belgium;eAgilent Technologies, Paris, France;fLaboratory of Molecular Parasitology, IBMM, Faculty of Sciences, Université Libre de Bruxelles, Gosselies, Belgium |
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Abstract: | Proteomic applications have been increasingly used to study posttranslational modifications of proteins (PTMs). For the purpose of identifying and localizing specific but unknown PTMs on huge proteins, improving their sequence coverage is fundamental. Using liquid chromatography coupled to mass spectrometry (LC–MS/MS), peptide mapping of the native apolipoprotein-B-100 was performed to further document the effects of oxidation. Apolipoprotein-B-100 is the main protein of low-density lipoprotein particles and its oxidation could play a role in atherogenesis. Because it is one of the largest human proteins, the sequence recovery rate of apolipoprotein-B-100 only reached 1% when conventional analysis parameters were used. The different steps of the peptide mapping process—from protein treatment to data analysis—were therefore reappraised and optimized. These optimizations allowed a protein sequence recovery rate of 79%, a rate which has never been achieved previously for such a large human protein. The key points for improving peptide mapping were optimization of the data analysis software; peptide separation by LC; sample preparation; and MS acquisition. The new protocol has allowed us to increase by a factor of 4 the detection of modified peptides in apolipoprotein-B-100. This approach could easily be transferred to any study of PTMs using LC–MS/MS. |
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Keywords: | LDL Mapping Q-TOF Recovery Apolipoprotein-B-100 |
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